ABCC9-related intellectual disability and myopathy syndrome (AIMS) arises from loss-of-function (LoF) mutations in the ABCC9 gene, which encodes the SUR2 subunit of ATP-sensitive potassium (KATP ) channels. KATP channels are found throughout the cardiovascular system and skeletal muscle and couple cellular metabolism to excitability. AIMS individuals show fatigability, muscle spasms, and cardiac dysfunction. We found reduced exercise performance in mouse models of AIMS harboring premature stop codons in ABCC9. Given the roles of KATP channels in all muscles, we sought to determine how myopathy arises using tissue-selective suppression of KATP and found that LoF in skeletal muscle, specifically, underlies myopathy. In isolated muscle, SUR2 LoF results in abnormal generation of unstimulated forces, potentially explaining painful spasms in AIMS. We sought to determine whether excessive Ca2+ influx through CaV 1.1 channels was responsible for myopathology but found that the Ca2+ channel blocker verapamil unexpectedly resulted in premature death of AIMS mice and that rendering CaV 1.1 channels nonpermeable by mutation failed to reverse pathology; results which caution against the use of calcium channel blockers in AIMS.

The skeletal muscle dihydropyridine receptor (DHPR) β1a subunit is indispensable for full trafficking of DHPRs into triadic junctions (i.e., the close apposition of transverse tubules and sarcoplasmic reticulum [SR]), facilitation of DHPRα1S voltage sensing, and arrangement of DHPRs into tetrads as a consequence of their interaction with ryanodine receptor (RyR1) homotetramers. These three features are obligatory for skeletal muscle excitation–contraction (EC) coupling. Previously, we showed that all four vertebrate β isoforms (β1–β4) facilitate α1S triad targeting and, except for β3, fully enable DHPRα1S voltage sensing [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. Consequently, β3 failed to restore EC coupling despite the fact that both β3 and β1a restore tetrads. Thus, all β-subunits are able to restore triad targeting, but only β1a restores both tetrads and proper DHPR–RyR1 coupling [Dayal et al., Proc. Natl. Acad. Sci. U.S.A. 110, 7488–7493 (2013)]. To investigate the molecular region(s) of β1a responsible for the tetradic arrangement of DHPRs and thus DHPR–RyR1 coupling, we expressed loss- and gain-of-function chimeras between β1a and β4, with systematically swapped domains in zebrafish strain relaxed (β1-null) for patch clamp, cytoplasmic Ca2+ transients, motility, and freeze-fracture electron microscopy. β1a/β4 chimeras with either N terminus, SH3, HOOK, or GK domain derived from β4 showed complete restoration of SR Ca2+ release. However, chimera β1a/β4(C) with β4 C terminus produced significantly reduced cytoplasmic Ca2+ transients. Conversely, gain-of-function chimera β4/β1a(C) with β1a C terminus completely restored cytoplasmic Ca2+ transients, DHPR tetrads, and motility. Furthermore, we found that the nonconserved, distal C terminus of β1a plays a pivotal role in reconstitution of DHPR tetrads and thus allosteric DHPR–RyR1 interaction, essential for skeletal muscle EC coupling.

Skeletal muscle excitation-contraction (EC) coupling roots in Ca2+-influx-independent inter-channel signaling between the sarcolemmal dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1) in the sarcoplasmic reticulum. Although DHPR Ca2+ influx is irrelevant for EC coupling, its putative role in other muscle-physiological and developmental pathways was recently examined using two distinct genetically engineered mouse models carrying Ca2+ non-conducting DHPRs: DHPR(N617D) (Dayal et al., 2017) and DHPR(E1014K) (Lee et al., 2015). Surprisingly, despite complete block of DHPR Ca2+-conductance, histological, biochemical, and physiological results obtained from these two models were contradictory. Here, we characterize the permeability and selectivity properties and henceforth the mechanism of Ca2+ non-conductance of DHPR(N617). Our results reveal that only mutant DHPR(N617D) with atypical high-affinity Ca2+ pore-binding is tight for physiologically relevant monovalent cations like Na+ and K+. Consequently, we propose a molecular model of cooperativity between two ion selectivity rings formed by negatively charged residues in the DHPR pore region.

In addition to the hallmark muscle stiffness, patients with recessive myotonia congenita (Becker disease) experience debilitating bouts of transient weakness that remain poorly understood despite years of study. We made intracellular recordings from muscle of both genetic and pharmacologic mouse models of Becker disease to identify the mechanism underlying transient weakness. Our recordings reveal transient depolarizations (plateau potentials) of the membrane potential to −25 to −35 mV in the genetic and pharmacologic models of Becker disease. Both Na + and Ca 2+ currents contribute to plateau potentials. Na + persistent inward current (NaPIC) through Naγ1.4 channels is the key trigger of plateau potentials and current through Ca v 1.1 Ca 2+ channels contributes to the duration of the plateau. Inhibiting NaPIC with ranolazine prevents the development of plateau potentials and eliminates transient weakness in vivo. These data suggest that targeting NaPIC may be an effective treatment to prevent transient weakness in myotonia congenita. Impact Statement Transient weakness in myotonia congenita is caused by depolarization secondary to activation of persistent Na + current in skeletal muscle.

In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRα1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.

The Ca 2+ -activated Cl − channel (CaCC) TMEM16A/Anoctamin 1 (ANO1) is expressed in gastrointestinal epithelia and smooth muscle cells where it mediates secretion and intestinal motility. However, ANO1 Cl − conductance has never been reported to play a role in skeletal muscle. Here we show that ANO1 is robustly expressed in the highly evolved skeletal musculature of the euteleost species zebrafish. We characterised ANO1 as bonafide CaCC which is activated close to maximum by Ca 2+ ions released from the SR during excitation-contraction (EC) coupling. Consequently, our study addressed the question about the physiological advantage of implementation of ANO1 into the euteleost skeletal-muscle EC coupling machinery. Our results reveal that Cl − influx through ANO1 plays an essential role in restricting the width of skeletal-muscle action potentials (APs) by accelerating the repolarisation phase. Resulting slimmer APs enable higher AP-frequencies and apparently tighter controlled, faster and stronger muscle contractions, crucial for high speed movements.

Formation of synapses between motor neurons and muscles is initiated by clustering of acetylcholine receptors (AChRs) in the center of muscle fibers prior to nerve arrival. This AChR patterning is considered to be critically dependent on calcium influx through L-type channels (Ca v 1.1). Using a genetic approach in mice, we demonstrate here that either the L-type calcium currents (LTCCs) or sarcoplasmic reticulum (SR) calcium release is necessary and sufficient to regulate AChR clustering at the onset of neuromuscular junction (NMJ) development. The combined lack of both calcium signals results in loss of AChR patterning and excessive nerve branching. In the absence of SR calcium release, the severity of synapse formation defects inversely correlates with the magnitude of LTCCs. These findings highlight the importance of activity-dependent calcium signaling in early neuromuscular junction formation and indicate that both LTCC and SR calcium release individually support proper innervation of muscle by regulating AChR patterning and motor axon outgrowth.

The L-type Ca2+ channel or dihydropyridine receptor (DHPR) in vertebrate skeletal muscle is responsible for sensing sarcolemmal depolarizations and transducing this signal to the sarcoplasmic Ca2+ release channel RyR1 via conformational coupling to initiate muscle contraction. During this excitation-contraction (EC) coupling process there is a slow Ca2+ current through the mammalian DHPR which is fully missing in euteleost fishes. In contrast to ancestral evolutionary stages where skeletal muscle EC coupling is still depended on Ca2+-induced Ca2+-release (CICR), it is possible that the DHPR Ca2+ conductivity during mammalian (conformational) EC coupling was retained as an evolutionary remnant (vestigiality). Here, we wanted to test the hypothesis that due to the lack of evolutionary pressure in post-CICR species skeletal muscle DHPR Ca2+ conductivity gradually reduced as evolution progressed. Interestingly, we identified that the DHPR of the early ray-finned fish sterlet (Acipenser ruthenus) is phylogenetically positioned above the mammalian rabbit DHPR which retained robust Ca2+ conductivity, but below the euteleost zebrafish DHPR which completely lost Ca2+ conductivity. Remarkably, our results revealed that sterlet DHPR still retained the Ca2+ conductivity but currents are significantly reduced compared to rabbit. This decrease is due to lower DHPR membrane expression similar to zebrafish, as well as due to reduced channel open probability (Po). In both these fish species the lower DHPR expression density is partially compensated by higher efficacy of DHPR-RyR1 coupling. The complete loss of Po in zebrafish and other euteleost species was presumably based on the teleost specific 3rd round of genome duplication (Ts3R). Ts3R headed into the appearance of two skeletal muscle DHPR isoforms which finally, together with the radiation of the euteleost clade, fully lost the Po.

Skeletal muscle excitation–contraction (EC) coupling is initiated by sarcolemmal depolarization, which is translated into a conformational change of the dihydropyridine receptor (DHPR), which in turn activates sarcoplasmic reticulum (SR) Ca 2+ release to trigger muscle contraction. During EC coupling, the mammalian DHPR embraces functional duality, as voltage sensor and l -type Ca 2+ channel. Although its unique role as voltage sensor for conformational EC coupling is firmly established, the conventional function as Ca 2+ channel is still enigmatic. Here we show that Ca 2+ influx via DHPR is not necessary for muscle performance by generating a knock-in mouse where DHPR-mediated Ca 2+ influx is eliminated. Homozygous knock-in mice display SR Ca 2+ release, locomotor activity, motor coordination, muscle strength and susceptibility to fatigue comparable to wild-type controls, without any compensatory regulation of multiple key proteins of the EC coupling machinery and Ca 2+ homeostasis. These findings support the hypothesis that the DHPR-mediated Ca 2+ influx in mammalian skeletal muscle is an evolutionary remnant.

Alternative splicing of the skeletal muscle CaV1.1 voltage-gated calcium channel gives rise to two channel variants with very different gating properties. The currents of both channels activate slowly; however, insertion of exon 29 in the adult splice variant CaV1.1a causes an ∼30-mV right shift in the voltage dependence of activation. Existing evidence suggests that the S3–S4 linker in repeat IV (containing exon 29) regulates voltage sensitivity in this voltage-sensing domain (VSD) by modulating interactions between the adjacent transmembrane segments IVS3 and IVS4. However, activation kinetics are thought to be determined by corresponding structures in repeat I. Here, we use patch-clamp analysis of dysgenic (CaV1.1 null) myotubes reconstituted with CaV1.1 mutants and chimeras to identify the specific roles of these regions in regulating channel gating properties. Using site-directed mutagenesis, we demonstrate that the structure and/or hydrophobicity of the IVS3–S4 linker is critical for regulating voltage sensitivity in the IV VSD, but by itself cannot modulate voltage sensitivity in the I VSD. Swapping sequence domains between the I and the IV VSDs reveals that IVS4 plus the IVS3–S4 linker is sufficient to confer CaV1.1a-like voltage dependence to the I VSD and that the IS3–S4 linker plus IS4 is sufficient to transfer CaV1.1e-like voltage dependence to the IV VSD. Any mismatch of transmembrane helices S3 and S4 from the I and IV VSDs causes a right shift of voltage sensitivity, indicating that regulation of voltage sensitivity by the IVS3–S4 linker requires specific interaction of IVS4 with its corresponding IVS3 segment. In contrast, slow current kinetics are perturbed by any heterologous sequences inserted into the I VSD and cannot be transferred by moving VSD I sequences to VSD IV. Thus, CaV1.1 calcium channels are organized in a modular manner, and control of voltage sensitivity and activation kinetics is accomplished by specific molecular mechanisms within the IV and I VSDs, respectively.

Ca2+-activated Cl- channels (CaCC) are expressed in various tissues and play important roles in numerous physiological functions such as epithelial secretion, olfactory and sensory transduction, cardiac excitability, and smooth muscle contraction. Even though CaCC mRNA was identified in human skeletal muscle (Huang et al., 2006), no CaCC conductance has been reported till date. Surprisingly, we found robust Ca2+-activated Cl- currents with current amplitudes of >100pA/pF at +80mV membrane potential in zebrafish skeletal muscle cells. Immunocytochemistry and subtype-specific CaCC current blockers allowed us to identify Ano1 (TMEM16A) as the channel protein responsible for this massive Cl- influx in zebrafish skeletal myotube. Whole-cell patch-clamp recordings revealed that this CaCC current is outwardly rectifying at sub-maximal Ca2+ levels and shows a linear current-voltage relationship at high [Ca2+]. Interestingly, this robust CaCC current can only be observed in wild-type zebrafish myotubes, which display intact SR Ca2+ release during excitation-contraction (EC) coupling. In contrast, the CaV1.1 β1-null zebrafish mutant relaxed, lacking the SR Ca2+ release, displayed no CaCC current. Thus, the CaCC current through Ano1 is activated by SR Ca2+ release during EC coupling. CaCC activation during EC coupling is close to maximum regarding Cl- influx, as seen in Ca2+-dependence experiments. Furthermore, we observed different CaCC current properties in the superficial slow and deep fast skeletal musculature. Further studies, to test if the CaCC current in zebrafish skeletal muscle plays a role in shaping the action potential by shortening the repolarization phase and thus allowing faster muscle contraction, are on the way. Supported by FWF P23229 and W1101-B12

In contrast to cardiac excitation-contraction coupling (ECC), skeletal-muscle ECC is based on Ca2+-influx-independent inter-channel communications between the dihydropyridine receptor (DHPR) and the ryanodine receptor (RyR1). The role of the small Ca2+ influx through the DHPR in mammalian skeletal muscle, which is not (immediately) required for ECC, is still enigmatic. Previously, we discovered that zebrafish, as well as all higher teleost fish, lack DHPR Ca2+ conductivity in skeletal muscle (Schredelseker et al., PNAS, 2010). Point mutation N617D in pore loop II of zebrafish DHPRα1S-b explained non-conductivity in fast muscle. To investigate the fascinating skeletal muscle DHPR Ca2+-conductivity / non-conductivity phenomenon we generated a non-conducting-DHPR knock-in mouse (n.c.DHPR) by introducing the N→D mutation into gene CACNA1S. Interestingly, homozygous n.c.DHPR mice are viable, fertile, visually indistinguishable, and identical in body-weight development to wild-type (WT) siblings. Myotubes isolated from newborn n.c.DHPR mice display complete lack of DHPR Ca2+ influx without altered ECC. No difference in locomotor activity (home cage activity), motor coordination (rotarod, beam walking), and muscle strength (endurance test, wire hang test) is observed in 3-7 months-old homozygous n.c.DHPR mice compared to WT. Identical results were obtained from forced frequency and fatigue tests on isolated EDL (fast twitch) and soleus (slow twitch) muscle fibers. As soon feasible, tests will be repeated on aged (18 months-old) mice to test for putative age-related accumulative effects on muscle performance, in order to understand if DHPR Ca2+ influx in mammalian skeletal muscle is a physiological necessity or just a tolerated evolutionary remnant of the ancestral pure Ca2+-influx dependent ECC of early chordates and phylogenetic branches below. Supported: FWF P-23229-B09; DK-W1101-B12.

The dihydropyridine receptor (DHPR) β1a subunit is crucial for enhancement of DHPR triad expression, assembly of DHPRs in tetrads, and elicitation of DHPRα1S charge movement--the three prerequisites of skeletal muscle excitation-contraction coupling. Despite the ability to fully target α1S into triadic junctions and tetradic arrays, the neuronal isoform β3 was unable to restore considerable charge movement (measure of α1S voltage sensing) upon expression in β1-null zebrafish relaxed myotubes, unlike the other three vertebrate β-isoforms (β1a, β2a, and β4). Thus, we used β3 for chimerization with β1a to investigate whether any of the five distinct molecular regions of β1a is dominantly involved in inducing the voltage-sensing function of α1S. Surprisingly, systematic domain swapping between β1a and β3 revealed a pivotal role of the src homology 3 (SH3) domain and C terminus of β1a in charge movement restoration. More interestingly, β1a SH3 domain and C terminus, when simultaneously engineered into β3 sequence background, were able to fully restore charge movement together with proper intracellular Ca(2+) release, suggesting cooperativity of these two domains in induction of the α1S voltage-sensing function in skeletal muscle excitation-contraction coupling. Furthermore, substitution of a proline by alanine in the putative SH3-binding polyproline motif in the proximal C terminus of β1a (also of β2a and β4) fully obstructed α1S charge movement. Consequently, we postulate a model according to which β subunits, probably via the SH3-C-terminal polyproline interaction, adapt a discrete conformation required to modify the α1S conformation apt for voltage sensing in skeletal muscle.

The CaV1.1 calcium channel is the voltage sensor of skeletal muscle excitation-contraction (EC) coupling. The current of the adult CaV1.1a splice variant is slowly activating, small and has poor voltage-sensitivity, whereas that of the embryonic CaV1.1e splice variant, lacking exon 29 in the IVS3-S4 linker, has an 8-fold higher amplitude, activates fast and at 30mV less depolarizing potentials (Tuluc et al.,2009). Here we created intra-molecular chimeras to test the hypothesis that the voltage sensors of homologous repeats I to IV differentially control current kinetics, voltage-dependence, and EC-coupling. Inserting the IVS3-S4 linker (mainly coded by exon 29) plus IVS4 into the corresponding region of repeat I fully restored CaV1.1a amplitude and voltage-sensitivity to CaV1.1e. Transferring the IVS3-S4 linker plus IVS4 into the homologous region of repeat II enhanced the voltage-sensitivity of the calcium current but fully abolished skeletal muscle EC-coupling. Any substitution in IS3-S4 resulted in accelerated activation-kinetics. However, slow activation-kinetics could not be transferred from the Ist to the IVth repeat. Interestingly, inserting IS3 plus the IS3-S4 linker into repeat IV fully restored the poor CaV1.1a voltage-dependence and small amplitude in the absence of exon 29. Secondary structure prediction revealed a beta-sheet in exon 29 that is missing in CaV1.1e. Point mutations which abolish this beta-sheet partially recapitulate the effects of deleting exon 29. Together these findings suggest a model according to which the voltage sensor of repeat IV controls the voltage-dependence and amplitude and its properties can be transferred to repeat I; the voltage sensor of repeat I determines the current kinetics exclusively in the context of this repeat; and the voltage sensor of repeat II controls EC-coupling. Funded by the Austrian Science Fund P20059-B05, P23479-B19 and MFI-2007-417.

The Ca2+ channel CaV1.1 is the voltage sensor of skeletal muscle excitation-contraction coupling. The classical skeletal muscle CaV1.1 isoform has poor voltage sensitivity and conducts a small, slowly activating Ca2+ current. In contrast, a splice variant lacking exon 29 (α1S-ΔE29) (Tuluc et al.,2009) has an 8-fold higher current amplitude, fast activation-kinetics, and a 30mV left-shifted voltage-dependence of activation. Therefore, the extracellular loop in repeat IV (IVS3-IVS4) mainly coded by exon 29 is a critical determinant of the characteristic gating properties of CaV1.1.

In the DHPRβ1-null zebrafish strain relaxed the lack of β1a results in reduced DHPRα1S membrane expression, in impediment of tetrad formation, and also in the elimination of α1S charge movement (Schredelseker et al., 2005, PNAS). Recently we postulated a model describing the β1a subunit as an allosteric modifier of proper α1S conformation (Schredelseker/Dayal et al., 2009, JBC) and thus enabling full DHPR functionality in skeletal muscle excitation-contraction (EC) coupling. To investigate if distinct regions of β1a might be responsible for inducing the voltage-sensing function of the DHPR, we expressed different β isoforms and chimeras in isolated myotubes of relaxed larvae for patch clamp (charge movement) analysis. Quantitative immunocytochemical analyses showed that all four β isoforms (β1-β4) were able to fully target α1S into triads. Interestingly, despite full triad targeting, β3 was unable to restore considerable charge movement (Qmax, 2.53 ± 0.50 nC/μF) in contrast to the other β isoforms (Qmax, 8.86 ± 0.93 to 9.94 ± 2.06 nC/μF) upon expression in relaxed myotubes. Systematic exchanges of variable regions and conserved domains of β1a with corresponding β3 sequences revealed significantly reduced Qmax restoration with SH3 and C-terminal chimeras (Qmax, 4.02 ± 0.28 and 5.57 ± 0.74 nC/μF, respectively). In contrast, β1a/β3 chimeras with the N-terminus, HOOK and GK domain exchanged showed complete restoration of charge movement. Together, our data suggest an essential role of the conserved SH3 domain and the variable C-terminus of β1a in the induction of the voltage-sensing function of the DHPRα1S in skeletal muscle EC coupling. Grants: FWF-DK-W1101-B12, FWF-P23299-B09

To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca2+channel (CaV) α1S-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of CaVα1Sin all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged α1ST1354S in dysgenic (α1S-null) myotubes. Whole cell patch-clamp analysis revealed that α1ST1354S produced significantly faster activation of L-type Ca2+currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α1S(α1SWT). In addition, α1ST1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca2+release and to larger action-potential-induced intracellular Ca2+transients under low (≤2 mM) caffeine concentrations compared with α1SWT. Thus our data suggest that an additional influx of Ca2+due to faster activation of the α1ST1354S L-type Ca2+current, in concert with higher caffeine sensitivity of Ca2+release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.

Antonella Pirone, Johann Schredelseker, Petronel Tuluc, Elvira Gravino, Giuliana Fortunato, Bernhard E. Flucher, Antonella Carsana, Francesco Salvatore, and Manfred Grabner Department of Medical Genetics, Molecular and Clinical Pharmacology, and Department of Physiology and Medical Physics, Innsbruck Medical University, Innsbruck, Austria; Dipartimento di Biochimica e Biotecnologie Mediche and Dipartimento di Scienze Chirurgiche, Anestesiologiche, Rianimatorie e dell’Emergenza, Università di Napoli “Federico II,” Napoli, Italy; CEINGE–Biotecnologie Avanzate, Napoli, Italy; and Istituto Di Ricovero e Cura a Carattere Scientifico– Fondazione SDN, Napoli, Italy